There has been a growing recognition that microfluidic devices have a wide range of applicability in the areas of biotechnology, genetic research, DNA diagnostics and thermocycling for carrying out temperature controlled processes. Of particular applicability is the amplification of DNA sequences through polymerase chain reactions (PCR). PCR replicates small amounts of DNA in a series of heating and cooling cycles and has been used in diverse research applications including biology, DNA sequencing, cloning, research, and genetic synthesis using published DNA sequences. Microfluidic devices also provide means for monitoring and controlling a wide variety of process parameters using resistivity and/or conductivity measurements.
Microfluidic systems comprise microfluidic devices or “chips” that have channels that are generally fabricated at the microscale, that is, having at least one channel cross sectional dimension (e.g., channel depth, width, or radius) of less than 1 mm, and typically in the range of from about 0.1 micrometers to about 500 micrometers. Planar chip technology employed in fabricating such devices is disclosed in Manz et al., Trends in Analytical Chemistry (1990) 10(5):144-149 and Manz et al., Advances in Chromatography (1993) 33:1-66. These references describe the fabrication of microfluidic devices and particularly microcapillary devices composed of silicon and glass substrates. It is well known that such devices can be employed for carrying out capillary electrophoresis, liquid chromatography, flow injection analysis, chemical reactions and synthesis.
Not surprisingly, chemical and biological analyses carried out in microfluidic devices require precise control over process parameters and, specifically, process temperatures. Biological reactions, as well as chemical reactions, generally, are exceedingly temperature sensitive requiring not only the ability to rapidly change processing temperatures during various stages of the chemical or biological processes but further require temperature uniformity from microchannel to microchannel. However, providing such temperature control and uniformity has proven to be a formidable challenge that those involved in this technology have yet to fully resolve.
Commonly, electrical energy has been employed to heat fluids contained within microfluidic channels. For example, in U.S. Pat. No. 5,965,410, the disclosure of which is incorporated herein by reference, electric current is applied through the fluids themselves. This technique has been employed successfully for a wide variety of chemical and biological applications, such as PCR. This global strategy can be fine tuned by directing electrical current through only portions of fluid-filled microchannels in order to selectively elevate temperature as processing parameters dictate.
In addition to the “Joule” heating described in referenced U.S. Pat. No. 5,965,410, fluid heating can be carried out by employing conventional heating mechanisms including the use of external heating elements such as hot plates or Peltier devices placed adjacent to the microfluidic channels to cycle the temperature of fluids contained therein. In addition, as described in co-pending U.S. application Ser. No. 10/123,100, resistive heaters in the form of longitudinally extending metallic filaments can be fabricated on the surface of a microfluidic device adjacent to the various microfluidic channels. As an electric current passes through the longitudinally extending metal films, heat is generated which is transferred directly to fluids contained within nearby microfluidic channels.
In order to gain further appreciation of microfluidic devices of the type referred to herein, reference is made to FIG. 1. FIG. 1 is a schematic example of a microfluidic channel network including body structure 2 that includes channel network 4 disposed therein. The microfluidic device also includes external sample accession capillary element or pipettor (not shown) that enters the device through an interface 6. The pipettor extends from the body of the microfluidic device so that materials can be brought into the channel network from sources external to the device itself, for example, from multiwell plates.
Channel network 4 also includes common channel 10 that receives materials drawn into the network from the pipettor element. This common channel is fluidly connected to a plurality of separate analysis channels 12-26. The analysis channels are used to perform different assays on separate aliquots of the sample material drawn into the sample network. The number of different analysis channels typically depends upon the desired rate of throughput for the overall system, and for each channel network incorporated in that system. Typically, a given channel network will include between about 1 and 20 separate analysis channels, and preferably between 5 and 15, with 8 to 12 analysis channels being most preferred.
Continuing reference to FIG. 1, each analysis channel typically is fluidly connected to a source of reagents, for example, reservoir 28 that may include either locus or patient specific reagents. Each analysis channel typically includes at least one, and often several, heating zones, for example, zones 26a and 26b, for carrying out different desired operations within the analysis channel. By way of example, within region 26a, an amplification reaction is optionally carried out to amplify the section of the patient's genomic DNA that includes the particular polymorphic locus. This is generally accomplished by combining the patient's DNA with appropriate amplification reagents, for example, primers, polymerase and dNTPs, followed by thermally cycling the contents of the channel, for example, within region 26a, through melting, annealing and extension processes, until sufficient amplified product has been produced.
As noted above, heating the fluid contained with the channels passing through region 26a can be carried out using electrical current supplied by electrodes in electrical contact with opposite ends of a suitable heating agent, such as the longitudinally extending metal films 30 (only one designated), or the fluid in the channel in the case of Joule-heating. Heat is then generated by applying current through the metal films 30 or the fluid in region 26a until the fluid in the channels in that region reaches the desired temperature. The process of Joule heating is described in detail in U.S. Pat. No. 5,965,410. Examples of metal films used as resistive heaters include those described in U.S. Pat. No. 6,132,580, the disclosure of which is incorporated by reference herein. Alternatively, conventional heat mechanisms may be employed, including the use of an external heating element, for example, a hot plate or a Peltier device, placed adjacent to the heating region to cycle the temperature therein.
FIG. 1 illustrates one embodiment of the use of resistive heaters for temperature control of multiple analysis channels. The resistive heaters can comprise multiple thin resistive metal films, shown as dotted lines, for example, 30, deposited on both sides of each analysis channel in region 26a. The resistive heaters are connected to electrical leads for the application of a voltage across the metal film. Current applied through the metal film heats the contents of the channels disposed therebetween. Temperature sensors can be incorporated into devices in accordance with the invention for measuring temperature within the heated region of the channel network. In the embodiment shown in FIG. 1, the temperature sensors comprise resistance thermometers that include material having an electrical resistance proportional to the temperature of the fluids contained within the microchannels.
In addition to the need to apply controlled amounts of energy to fluids contained within microchannels in order to elevate their temperatures, it is also necessary to provide means to cool such fluids to further control processing conditions. For example, reference was made previously to the use of such devices in carrying out PCR cycling. Such cycling requires the steps of denaturation, primer annealing and DNA synthesis. During denaturation, the starting mixture is first heated to about 95° C. for separating the double strands of DNA. After denaturation of the DNA, the mixture is cooled to about 55° C. to allow the primers to bind to their complimentary sequences on separated strands. Thereupon, the mixture is heated to a temperature of about 72° C. so that the DNA polymerase catalyzes the extension of the annealed primers on the template strand.
Although commercially available apparatus has been employed in carrying out the PCR cycling protocol, microfluidic devices such as those shown in FIG. 1 are particularly well adapted for doing so. However, again, PCR cycling requires exacting precision to uniformly and accurately raise and lower the temperature of the fluids contained within the subject microchannels as processing conditions so dictate. Furthermore, it is advantageous for the transitions in temperature between the temperatures required for denaturation, primer annealing and DNA synthesis occur as rapidly as possible.
It is intuitively obvious that in employing a microfluidic device such as that shown in FIG. 1 having parallel processing channels employed in carrying out, for example, in-line PCR manipulation, every reaction channel must produce equivalent thermal profiles. However, the tendency has been to increase channel density within such microfluidic devices, which, in turn, increases the amount of power delivered to such devices. Without adequate removal of heat, “hot spots” on the microfluidic chips can form resulting in thermal gradients between reaction channels. In this regard, reference is made to FIG. 2, which graphically displays the normalized average temperature for nine parallel microfluidic channels that are equally spaced, like the eight parallel channels in region 26a of FIG. 1, and heated by Joule heating. The temperature is scaled such that the collective average temperature Tave of all nine channels equals one. Significant temperature differences between channels become measurable, particularly as channel-to-channel spacing is reduced. This is not particularly surprising since one channel would have a tendency to transfer energy to adjacent parallel channels, particularly as spacing between channels diminishes. The channels on the edges of the group of parallel channels, such as the channel numbers 1 and 9, tend to be cooler than the interior channels because those outer channels only have one adjacent channel. The temperature of the interior channels even varies, with interior channels closer to the edge tending to be cooler. Variation of channel temperatures can be dramatic, as much as 30° C. across the various parallel microchannels when operating at set points of 95° C. Although this effect can be diminished by using individually controllable power supplies to heat each channel, the resulting complexity in equipment and fabrication costs make such an approach undesirable.
It is also of critical importance that any such microfluidic device possesses the ability to controllably remove thermal energy from the device and thus fluids contained therein. For example, in using such devices to carry out PCR reactions, it is necessary that fluids contained within the microchannels be maintained at temperatures of approximately 95° C., 72° C. and 60° C., so some of the temperature transitions require removal of heat from the microfluidic device. The present invention teaches techniques for doing so, including applying fluids directly against the microfluidic chip surfaces, which can be employed to effectively introduce and withdraw thermal energy. When used in conjunction with other aspects of the present invention, the details of which will be disclosed hereinafter, one is able to achieve a device capable of microfluidic manipulation with a degree of temperature control that has heretofore been unavailable.
It is thus an object of the present invention to provide a microfluidic device capable of controlling thermal energy applied to and withdrawn from fluids being manipulated therein.
It is yet a further object of the present invention to provide a microfluidic device having multiple channels for carrying fluids therein in which the thermal energy and thus temperature within each channel is capable of being controlled and maintained consistent with temperatures of fluids in companion channels.
These and further objects will be more readily appreciated when considering the following disclosure and appended claims.